ABSTRACT Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.

中文翻译：

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纳米粒子介导的传递和小分子触发的细胞核中蛋白质的激活

摘要 蛋白质转染是研究或操纵细胞过程的通用工具，也显示出巨大的治疗潜力。然而，用于有效和最低限度细胞毒性递送的成本有效技术的曲目仍然有限。介孔二氧化硅纳米粒子 (MSN) 是一种多功能纳米载体，可用于多种分子的细胞递送，它们的合成简单且经济，并且在蛋白质递送方面显示出巨大的前景。在这项工作中，我们提出了优化向细胞核输送活性蛋白质的一般策略。我们生成了一个基于双分子金星的光学传感器，它专门检测活性和生物可利用的蛋白质，以实现蛋白质输送的多参数优化性能。结合细胞活力测试，我们最大限度地提高了 MSN 蛋白质递送和生物相容性，并实现了 80% 的高效蛋白质转染率。使用传感器测量活细胞蛋白质传递动力学，我们观察到细胞群内的异质时间，这可能对功能研究产生混淆影响。为了解决这个问题，我们将感兴趣的分裂或二聚化依赖性蛋白质与化学诱导的二聚化 (CID) 成分融合，从而允许在细胞递送后控制其活性。使用分裂的金星蛋白，我们直接表明添加小分子二聚体会导致整个细胞群中传递的蛋白质同步激活。